Since its first identification as a “red protein” in bovine milk more than 65 years ago, and its purification in 1960, lactoferrin has intrigued and puzzled researchers. Subsequent determination of its amino acid sequence, three dimensional structure and detailed iron binding properties firmly established lactoferrin is a glycoprotein, as a member of the transferrin family, and reinforced the natural presumption that its biological function related to iron binding.
Different research centers have played an important role stressing on some biological key functions of the protein. Lactoferrin was isolated as a major component in the specific granules of the polymorphonuclear leukocytes with an important role in the amplification of the inflammatory response. Extensive work by Masson and its Belgian group has established a clear role for lactoferrin in cellular immunity and has led to the identification of specific lactoferrin-receptors on macrophages, intermediation of endotoxic shock and hyposideremia. Pioneering efforts by Montreuil and his French group unraveled the biological chemistry of lactoferrin. Lönnerdal has opened the nutritional role for lactoferrin in the absorption of metals ions in the intestinal tract. Broxmeyer and his co-workers reported a regulatory function for lactoferrin in myelopoiesis. From his side, Reiter reported the ability of milk lactoferrin to inhibit the growth of some microorganisms and found that nutritional deprivation of the bacteria from iron accounted for the antimicrobial activity. Arnold and his collaborators reported bactericidal activity for lactoferrin against a variety of microorganisms. Tomita and his research group at Morinaga Milk Industry in Japan has found that acid/pepsin hydrolysis of lactoferrin could generate cationic antimicrobial peptides “lactoferricin”.
Several studies have established that lactoferrin supplementation could provide exceptional health benefits and a powerful protection against several illness. Functional characterization technologies have elucidated the molecular mechanisms of lactoferrin-mediated multifunctional activities. Furthermore, investigators from laboratories around the world have validated the functional outcomes with lactoferrin supplements in randomized human trials and in vivo experimental models.
But if the multifunctional activities of this extracellular glycoprotein that functions as a key component of the first line of mammalian immune defense against environmental insults have been demonstrated using a good quality of lactoferrin produced in the laboratory, we have discovered that it is not the case with the lactoferrin produced commercially.
During the industrial process, the Lf is extracted from milk or whey in presence of other Milk Basic Proteins (MBP) such as lactoperoxidase, some immunoglobulins and other contaminants of which the concentration is dependent of the specificity of the cationic ion exchange resin. It is an easy process that consists to extract and purify the Lf. In fact, we have the advantage that the most part of proteins and enzymes contained in the MBP are colored. The elution of the different components bound on the resin will be performed using solutions containing different NaCl concentrations. Using such procedures, the industrial producers consider that a purity between 90 to 92% correspond to a Lf enough pure to be used for the different applications.
However, none of these processes, nor any other existing process for commercial-scale purification of lactoferrin, are able to remove contaminants that affect the stability and activity of the lactoferrin.
It appears that contaminant enzymes are present in currently existing commercial lactoferrin preparation. These enzymes are co-purified during lactoferrin purification from milk or whey
Regarding the contaminants, as it will be demonstrated below, we have also found that the angiogenin can be purified during the purification of the Lf. This molecule has a molecular weight of 15 kDa and an isoelectric pH of 9.5 very close to the Lf.
This molecule is responsible to the creation of the blood vessel to feed the cancer cells, neo-vascularization indispensable to the growth of tumors and to the development of the metastasis. During the purification of the Lf, this molecule has been concentrated at least 4 times what is certainly not beneficial for the health of the consumers.
Angiogenin contributes to an inflammatory process that allows the transmigration of endothelial and smooth muscle cells through basement membrane to enter a site of injury. Angiogenin promote the neovascular of the tumor cells and promote the proliferation of the metastasis of the cancer cells.
Angiogenin is a protein of 15 kDa with an isoelectric pH of 9.5 that means very close to Lf. As described by Strydom et al., in 1997 (Eur. J. Biochem, 247, 535-544, angiogenin was applied to a CM-52 (cation-exchange chromatography resin) and was eluted with 1M NaCl in 50 mM sodium phosphate, pH 6.6 solution. So it is not surprising that this molecule is co-purified with Lf and has been detected in the SDS-PAGE gel in presence of all the commercial Lf.
Another problem is the production by thermal treatment of Lf polymers that we have also demonstrated, see below.
Therefore, there is a great need for new purification and stabilization methods of lactoferrin preparations in order to remove contaminants, the protein degradation and the LPS to enhance, the activity on bacterial growth and to preserve the protein stability, for a longer period of time.
Although originally identified as an abundant protein in milk secretion, lactoferrin is expressed predominantly by surface epithelia and secreted into the mucosal environment. As described, lactoferrin is produced at high levels not only in the milk but also in nasal and tracheal passages and in gastric, genital, and ophthalmic secretions. Lactoferrin is also produced at high levels in neutrophils where it is stored in secondary granules and released during inflammation and contribute to their antimicrobial activity.
Lactoferrin contains 2 homologous iron binding domains that sequester available iron and can deprive iron-requiring domains bacteria of this essential growth element. In this manner, the protein exerts a bacteriostatic effect against a large range of microorganisms and certain yeast. Moreover, lactoferrin, by the presence of its cationic peptide located close to the amino-terminus of the protein, has shown to possess both bactericidal and anti-endotoxin activities that are independent of the iron binding function of the protein. This region acts by disrupting bacterial membranes and by binding and inactivating bacterial lipopolysaccharides containing the lipid-A called also endotoxins (see FIG. 1)
Lactoferrin is also able to regulate cellular signaling pathways, which affect activities such as its alleviation of inflammation, promotion of bone growth and suppression of carcinogenesis.
Thus its anti-inflammatory activity is linked to an ability to inhibit the production of pro-inflammatory cytokines, but by several distinct mechanisms, and its regulation of bone growth that occurs through mitogen-activated protein kinase pathways. Increasing number of studies show that lactoferrin possesses anti-cancer properties, inhibiting the growth of cancer, that stem from its ability to modulate pathways that impinge on the cell cycle or result in upregulation of the expression of cytokines as interleukin-18.
Moreover the antimicrobial functionality of lactoferrin is dependent on its protein conformational, metal binding and milieu conditions (Naidu A S and Arnold R R., 1995, Lactoferrin interactions and biological Functions pp 259-275 Totowa, N.J., Humana Press). Antimicrobial activity is enhanced when lactoferrin binds to the microbial surfaces. The specific lactoferrin binding microbial targets have been identified on different Gram-positive and Gram-negative bacterial pathogens (Naidu S S et al., 1991, APMIS, 99, 1142-1150). The high-affinity interaction of lactoferrin with pore-forming outer-membrane proteins of Gram-negative enteric bacteria including Escherichia coli, is critical for the antimicrobial outcome of lactoferrin (Erdei et al., 1994, Infect Immun, 62, 1236-1240). Thus, lactoferrin-mediated outer-membrane damage in Gram-negative bacteria and the lactoferrin-induced antibiotic potentiation by causing altered outer membrane permeation are typical examples of such antimicrobial outcomes (Naidu et al., Diagn Microbiol Infect, 1988, Infect Immun, 56, 2774-2781). Lactoferrin interaction with the microbial surface, the outer membranes in particular, has led to other antimicrobial mechanisms such as microbial adhesion-blockage to intestinal epithelia and specific detachment of pathogens from gut mucosa. Specific binding of lactoferrin could instantly collapse bacterial outer membrane barrier function and leads to the shutdown of pathogen colonization factors and enterotoxin production.
From another side, Appelmelk and his collaborators (Appelmelk B J, et al., 1994, Infection and Immunity, 62, 2628-2632) have found that Lf binds to the lipid A, part of the LPS and Elass Elass-Rochart E, et al., 1995, Biochem J. 312, 839-845) has demonstrated that this binding site is located in the N-terminal (peptide 1 to 52) of the lactoferrin where are also located the main part of the activities of the Lf. From these results, it is easy to understand the relation existing between the activity of the lactoferrin and the presence of the LPS bound on the molecular structure of the lactoferrin.
There is a continuous transfer of LPS and endotoxin from the intestinal lumen into the bloodstream. In healthy individuals, plasma inactivates the intestinal influx of LPS and endotoxin and protects internal organs from damage. However, any disturbances in gut permeability could increase LPS and endotoxin transfer into the bloodstream. Such massive influx could exhaust the ability of plasma to inactivate LPS and endotoxins and ultimately lead to clinical endotoxemia (Opal S M, 2002, J. Endotoxin Res, 8, 473-476). Experimental evidence suggest that reactive oxygen species are important mediators of cellular injury during endotoxomia, either as result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. An important mechanism to prevent physiological endotoxemia is to reduce lipopolysaccharides (LPS) from the intestinal lumen.
On its N-terminal (lactoferricin peptide) Lf binds to lipid-A, the toxic moiety of LPS with high affinity and works as a therapeutic agent to neutralize effects of LPS and endotoxins (Appelmelk B J et al, 1994, Infect Immun, 62, 2628-2632). Lf could effectively reduce LPS and endotoxin influx into the bloodstream while toxins still are inside the intestinal lumen but to reach such result, it is important that Lf is manufactured free of LPS and endotoxin. Moreover, if the Lf feeds by the healthy person is covered by LPS, these LPS could be removed from the molecule and be transferred into the bloodstream.
In this process, however, Lf is also depleted rapidly and may not be present in sufficient amounts to perform this function if LPS and endotoxins are continuously released in large quantities (Caccavo et al, 2002, J. Endotoxin Res, 8, 403-417). A protective effect for Lf against lethal shock induced by intravenously administrated endotoxin has been reported. Lf-mediated protection against endotoxin (if the molecule is itself free of endotoxin during its production) challenge correlates with both-resistance to induction to hypothermia and an overall increase in wellness. In vitro studies with a flow cytometric measurement indicated that Lf inhibits endotoxin binding to monocytes in a dose-dependent manner, which suggests that the mechanism of Lf action in vivo could be due to the prevention of induction of monocyte/macrophage-derived inflammatory-toxic cytokines (Lee W J et al, 1998, Infect Immun, 66, 1421-1421).
Human clinical trials have also showed a positive influence of Lf consumption in primary activation of host defense (Yamauchi et al, 1998, Adv Exp Med Biol, 443, 261-265). Healthy people showed improvement in their serum neutrophil function including enhanced phagocytic activity and superoxide production. Furthermore, specific interaction of Lf with alveolar macrophages, monocytes, kupfer cells, liver endothelia, neutrophils, platelets, and T-lymphocytes emphasizes the role of Lf in mucosal and cellular immunity (Hanson L A, 1988, Biology of human milk. Nestlé Nutrition Workshop series, 15, New-York, Raven Press). Nevertheless, all this activity due to the interaction of the Lf with these cells is decreased by the presence of the LPS on the Lf structure and by the damages of the glycan chains of the Lf due to the use of the too high temperatures (>550° C. after 15 seconds) during the manufacturing process and due to the too high temperature for the drying of the molecule, and also by the presence of Lf polymers which appear during the heat treatment of the molecule.
Concerning gut maturation and mucosal repair it has been demonstrated that oral Lf administration could function as an immune stimulating factor in the intestinal mucosa.
The gastrointestinal tract matures more rapidly in the newborn during breast feeding. This activation is dependent on Lf binding to the intestinal epithelia. Lf could potentiate thymidine incorporation into crypt cell DNA in vivo. This trophic effect contributes to cell regeneration and tissue repair of intestinal mucosa in conditions such as gastroenteritis (Nichols et al., 1990, Pediatr Res., 27, 525-528). The presence of the LPS on the Lf structure decrease this activity of the Lf due to the fact that the Lf binding to the intestinal epithelia is located to the peptide (1-52) that means at the same place that the LPS
Lf also plays an important role in the intestinal absorption of iron and other trace essential elements such as zinc, copper (Lonnerdäl B., 1989, J. Nutr. Suppl, 119, 1839-1844). Lf also protects the gut mucosa from excess uptake of heavy metal ions. Specific Lf binding receptors in the human duodenal brush border are involved in the iron absorption (Cox et al 1979, Biochem Biophy Acta, 588, 120-128). An intestinal Lf receptor was identified. Increased iron absorption via this Lf receptor from the intestinal brush-border membranes have been reported (Kawakami H et al., 1991, Am. J. Physiol, 261, G841-G846 and Rosa G et al., J. Med. Biol. Res, 27, 1527-1531) and here also the Lf peptide which is responsible to the binding of the molecule to its specific receptor has been localized on the peptide 1-52 which is also responsible to the binding of the LPS.
Concerning its anti-tumor activity, Lf is shown to enhance natural killer (NK) activity of monocytes in a dose-dependent manner. Lf strongly increases both NK and lymphokine-activated killer (LAK) cell cytotoxic functions. Lf is an effective modulator of cell-mediated immune response and serum cytotoxic factors at low dosages if the LPS are not bound on Lf structure and if Lf is not contaminated by the angiogenin. However, at higher concentrations the Lf-mediated induction could lead to a positive or negative feedback according not necessary to the density and subsets of the immune cell population but also to the presence of the LPS on the Lf structure.
Discovery of specific Lf receptors on macrophages, T and B-lymphocytes and leukemia cells establish the potential anti-tumor potential of Lf (Shau et al., 1992, L. Leukoc Biol, 51, 343-349) which could be eliminate by the presence of the LPS on its structure.
The anti-inflammatory activity of the Lf is primarily associated with its ability to scavenge iron. It is known that accumulation of iron in inflammated tissues could lead to catalytic production of highly toxic free radicals. During an inflammatory response, neutrophils migrate to the challenge site to release their Lf containing acidic granules. This results in the creation of a strong acidic milieu at the inflamed tissue site to amplify iron-sequestering and detoxification capacities of Lf. Besides modulating iron homeostasis during inflammation, there is mounting evidence that Lf could directly regulate various inflammatory responses. This iron-independent mode of action is based on Lf binding to bacterial LPS, which is major pro-inflammatory mediator during bacterial infections and septic shock (Miyazawa et al., 1991, J. Immunol, 146, 723-729). Lf could play an important role in the modulation of gastric inflammation, since this protein is also expressed in the gastric mucosa of the stomach and interacts with receptors localized on gastric intestinal epithelial cells. This activity of the Lf is completely decreased or even eliminated when LPS cover the Lf structure. Several in vivo studies have shown that oral administration of Lf could reduce gastric induced by Helicobacter pillory and protect gut mucosal integrity during endotoxemia. Here also such activity of the Lf is very poor when the LPS are bound on the Lf structure.
The iron-independent activity of the Lf can be described as follows: One of the central proinflammatory functions of endothelial cells is the recruitment of circulating leukocytes at inflammatory tissue sites. Lipolysaccharides (LPS) or endotoxins is a predominant glycolipid in the outer membrane of Gram negative bacteria. The LPS are potent stimulators of inflammation that induce either directly or through the intermediary of cytokines, the expression of adhesions molecules such as endothelial-leukocyte adhesion molecule (E-selectin) and intracellular adhesion molecule 'ICAM-1). Endotoxin stimulation of endothelial cells is mediated by soluble protein found CD14 (sCD14), a specific receptor. CD14 is a 55 kDa glycoprotein that exists in the serum and as an anchored protein (mCD14) on the surface of monocytes-macrophages. In this mechanism, depending of the concentration of the LPS (endotoxins), there is the presence of an intermediate called the LPS-binding protein (LBP), which catalyses the transfer of LPS monomers from aggregates to CD14 to form a sCD14-LPS complex. Thus, the activation of endothelial cells by the sCD14-LPS complex or by the LPS alone causes various pathophysiological reactions including fever and hypotension, promotes leukocytes infiltration and microvascular thrombosis and contributes, during septic shock, to the pathogenesis of disseminated intravascular inflammation.
Nevertheless, lactoferrin found in exocrine secretions of mammals and released from granules of neutrophils during inflammation is able to modulate the activation of the cells and avoid the severe damages causing by the presence of the LPS.
Following infection, lactoferrin concentrations, higher than 20 μgr/ml, can be detected in blood. Lactoferrin is part of a primary defense system against the inflammation. Any presence of bacteria in the organism, is going to induce the inflammation, cancer and other pathologies. This induction is going to stimulate immune responses including cytokine production, increase of expression of cell adhesion molecules, and pro-inflammatory mediator secretion by monocytes, macrophages and neutrophils, which are into specific host tissues by systemic LPS exposure. The response of the host to LPS is mediated by immune modulator molecules such as tumor necrosis factor alpha (TNF-alpha), members of the interleukins (IL) family, reactive oxygen species, and lipids. Overproduction of those mediators induces tissue damage that precedes multiple organ failure.
Lactoferrin prevents the LBP-mediated binding of LPS to mCD14 and decreases the release of cytokines from LPS-stimulated monocytes. Lactoferrin might also modulate the inflammatory process. Indeed, studies reported the protective function of lactoferrin against sublethal doses of LPS in mice. In conclusion, the ability of lactoferrin to bind free LPS may account, in part, for the anti-inflammatory activities of the molecule.
It is the reason why when the human and the animal take orally or by injection lactoferrin to reinforce or to avoid the deficiencies of its primary defense system, it is primordial that the quality of the lactoferrin is identical to the one which is produced from the endogen way in the healthy human who has to protect himself against the microbial invasions. Knowing that during the aging process, the endogenic lactoferrin production becomes very poor, obliging the patients to take exogenic lactoferrin either orally or by injection.
Contaminants in source material could compromise the human health applications of Lf. Several factors including the origin of source material, protein purification, drying process and harvesting methods, manufacturing environment and storage conditions, all cumulatively contribute to the bioburden of Lf protein. Accordingly, when used as a source material, milk, whey or milk serum could carry through fermentative streptococci (Streptococcus thermophilus . . . ) and a medium with an acidic environment could selectively enrich several yeast and molds. Incidentally, these microbial populations are commonly known to proliferate and competitively limit the growth of several probiotics.
Lf derived from milk with a contamination of the milk pool from mastitis source could introduce the presence of LPS from gram-positive cocci including Streptococcus uberis, Staphycoccus aureus and coagulate-negative staphylococci. On the other hand, environmental contaminants such as spore-forming Bacillus spp, Acinetobacter calcoaceticus, Klebsiella oxytoca, Pseudomonas spp, and coliform including E. coli. and the LPS of such microorganisms could gain entry into Lf material through elution buffer, biofouled equipment, air ducts, etc. . . . . Similar microbiological quality issues could exist for the GMO-derived and recombinant Lf proteins from various expression such as rice, tobacco, yeast, cell cultures or transgeninc animals. Therefore, elimination or significant reduction of such LPS microbial contaminants is highly desirable for human health applications of commercial Lf, in general.
As it is explained here above, the LPS and endotoxin content in the source material could adversely affect the Lf applications. The lipopolysaccharides (LPS) in the gram-negative bacterial outer membrane typically consist of a hydrophobic domain known as lipid-A (or endotoxin), a non-repeating core oligosaccharide, and a distal polysaccharide (or O-antigen) (Erridge et al., 2002, Microbes Infect, 4, 837-851). LPS and endotoxins could stimulate the induction of cytokines and other mediators of inflammation, which in turn could trigger a broad range of adverse physiological responses (Raetz et al., 2002, Annu Rev Biochem, 71, 635-700). Gram-negative bacterial bioburden of milk or its derivatives used in protein isolation, processing plant environment and conditions cumulative contribute to LPS and endotoxin levels in an Lf source material. It has been reviewed the potential reservoirs for endotoxin contamination during isolation of protein materials (Majde et al., 1993, Peptides 14, 629-632). Rylanders (Rylander 2002, J. Endotoxin Res, 8, 241-252) has also reviewed the occurrence of endotoxin level in different environmental conditions and further pointed out the risks associated with non-bacterial endotoxins, particularly 1-3-β-D-glucan from mold cell walls. Thus, the microbial keeping standards of chromatographic resins, sanitation practices of processing equipment even more significantly the water quality used in Lf purification, could cumulatively contribute to the LPS and endotoxin levels of the purified Lf material and thereby could limit in vivo applications of commercial Lf. Pre-existence of Lf-LPS and endotoxin complexes reduce the potential of Lf interaction with gut epithelia and diminish its ability to control intestinal influx of LPS and endotoxins.
Then, all the commercial lactoferrin should have to be devoided of LPS bound on its molecular structure. For example, Ward, Loren and col. in WO2009/009706 have described a method to remove the endotoxins bound to Lf and to produce endotoxin-free lactoferrin product (EFL). That is not the case if you analyze the LPS concentration using the limulus test. It has been also demonstrated that when this concentration of LPS bound on the lactoferrin structure is too important, the complex LPS-Lf is able to induce production of inflammatory mediators in macrophages to some extent, rather than inhibit totally LPS activity. It is mainly due to the fact that when the LPS concentration is too important, there is an equilibrium between LPS bound→LPS-free and it is the presence of the LPS-free which induce production of inflammatory mediators. For safety, reasons that oblige the Lf producers to purify the molecule exempt of LPS bound on the surface of the Lf molecular structure.
Manufactured from the milk and/or the whey, it is normal that the lactoferrin is covered on its molecular structure by the bacterial LPS existing in the milk and that can be dangerous if such milk pool has been contaminated by microbial contaminated milk responsible of mastitis cows. We know that a part of the lactoferrin activities is represented by its anti-bactericidal role binding to the LPS of the bacteria existing in the milk. That means it is not surprising to extract from the milk a lactoferrin fully covered by LPS which has lost an important part of its biological activity regarding the antioxidant, the antibacterial and its activity to inhibit the bacterial biofilm formation and its prebiotic activity respectively.
Moreover Lf could be denaturized by heat treatment. There are different parameters that can be used to study the thermal stability of the lactoferrin. The heat treatment denaturation follows a first order kinetic. The denaturation increases with the temperature. The iron-free lactoferrin (Apo-Lactoferrin) shows a more rapid denaturation than the iron-saturated lactoferrin (Holo-Lactoferrin). That reflects to a more stable conformation when it is bound to iron. During thermal denaturation, the break of several binding provokes important changes in the Lf structure. The thermal stability is increasing in presence of other milk components due to the interaction between the lactoferrin and caseinates and other milk proteins.
The lactoferrin that is extracted from milk has an iron saturation level between 9 to 20% of the iron-saturated lactoferrin. However, after pasteurization of the milk or cheese whey, the protein which is extracted, has not the same activity level and not the same values compared to the lactoferrin, which has been extracted before any heat treatment of the milk or the cheese whey
In fact, the heat treatment is able to destroy the glycan chains of the molecule which are important to protect the lactoferrin against proteolytic enzymes that are present in the stomach and is also able to produced Lf polymers. This effect has been also demonstrated by the fact that when lactoferrin is submitted to a heat treatment, the molecule has a higher absorbance power at 280 nm (Table 1).
The destroying of the glycan chains, which are sensitive to the heat treatment will also increase the non-specific binding of the lactoferrin on the cells. Instead to promote the cell growth, the non-specific binding of the lactoferrin will rather induce the suffocation of the cells. Actually, it has been established by the producers of commercial Lf that the purity of Lf is determined by Reverse Phase HPLC using an acetonitril gradient. Analysing the purity of some commercial Lf, we observe that the proteic contaminants represent around 8 to 9% versus the Lf peak. Nevertheless, diluting the same amount of commercial Lf, adjusted by the ash and moisture content, we have not found the same optical density at 280 nm. That means that some proteins eluting as Lf can increase the optical density. In the FIG. 2 , , , we can observe that Lf analyzed by ion-exchange chromatography FPLC (Mono-S resin-Sulfopropyl) show a smaller surface compared to the heat-treatment Lf surface. The reduction of the height of the surface is due to the presence of a new surface which corresponds to the shoulder observed with the FPLC analysis and that we call peak C to simplify the description of the chromatogram. We could also observe that the Lf surface is split into two parts: peak A and peak B
very closed each other and corresponding for the peak A to the presence of one sialic acid content which give to the molecule a less basic behavior compared to the native one which does not contain sialic acid In case of the Lf-NFQ, the Lf surface is also composed of two surfaces (surface A and surface B). The shoulder (surface C) is only observed in the commercial Lf. The shoulder or surface C has a higher absorbance power at 280 nm compared to the native Lf see Table 1 below.
Anyway, we can consider that the peak A and peak B are parts of the pure Lf. The presence of the peak C cannot be detected with the use of the Reverse Phase chromatography. To understand the presence of this peak C, we have carried out the complete absorbance spectra from 280 nm to 800 nm and we have observed a band of Soret at 410 nm (FIG. 2) which is independent of the iron content in the Lf because this band of Soret should have to be present at a wavelength closed to the 465 nm. Moreover, the absorbance of this peak C at 280 nm is almost double to the Lf one.
Collecting only the peaks A and B, and applying again on the Mono S resin, we can notice that only the peaks A and B are present in the chromatogram without to be contaminated by the peak C. On the other hand, if we submitted the solution containing the peaks A and B to a temperature of 72° C. during 5 minutes and that we analyze this solution on the Mono S resin, we observe an important decrease of the surface of the peak A and of the surface of the peak B compared to the original chromatogram but also an appearance of the peak C (FIG. 2). More long time, we submit Lf to a heat treatment, more the peaks A and B will have a lower surface and more the peak C will be important.
If we compare on the Reverse Phase, the chromatogram of the Lf without heat treatment and the chromatogram of the same Lf but which has been submitted to a heat treatment (72° C.) during 5 min, we can notice that the surface of the Lf without heat treatment is lower that the surface of Lf having submitted a heat treatment (FIG. 3). The peak C has been characterized as Lf polymers having a much higher absorption power.
TABLE 1Absorbance at 280 nm for a Lf solutionHeating (30 seconds)of 1 mg/ml<50° C. 1.32670° C.1.3880° C.1.4285° C.1.42
The problem is not only based on the fact that the lactoferrin purified by the manufacturer has lost a percentage of its biological activities what could be compensate by the adding of an higher concentration of the molecule but by the fact than more we advise the use of a high Lf concentration to reach a certain level of activity, more we recommend the use of a high LPS concentration. That could automatically induce the inflammatory process instead to protect the patients (Table 3).
Several Lf products are currently available in the health food markets worldwide. A majority of such products are derived from partially isolated Lf from colostrums, milk or cheese whey. Furthermore, the microbiological and toxicological quality issues compromise the in vivo performance standards of Lf as a potent food material.
Lactoferrin is usually purified from milk or whey (milk whey or cheese whey) by one or more different types of chromatography resins such as ion exchange, especially cation-exchange, affinity (immobilized heparin, single strand DNA, lysine or arginine) dye affinity and size exclusion. Ultrafiltration membrane can also be used to separate lactoferrin from milk or whey. Tomita and his collaborators (Tomita et al., 2002, biochem Cell Biol, 80, 109-112) have given an example of the industrial process which uses both cation-exchange chromatography and tangential-flow membrane filtration. Other purification using cation-exchange chromatographies have been described by Okonogi and his co-workers (Okonoki et al., New Zealand Patent No 221,082), by Ulber (Ulber et al., 2001, Acta Biotechnol, 21, 27-34) and Zhang and his co-workers (Zhang et al., Milchwissenschaft 2002, 57, 614-617). Some researchers have used the hydrophobic properties of the molecule to purify the lactoferrin using hydrophobic interaction chromatography. Machold has described the retention behavior of lactoferrin on several hydrophobic interaction resins under range of salts concentrations (Machold et al, 2002, J. Chromatogr. A972, 3-19).
Different methods have been largely described by Dr Denis Petitclercq in the patent application WO 2006/119644 and the aim of his invention was to provide a process to remove enzyme contaminant responsible for lactoferrin degradation. The removal of these enzymes or addition of specific inhibitors would prevent degradation of a lactoferrin preparation and loss of activity of lactoferrin. He has applied his process to all commercial lactoferrin demonstrating that it is possible to improve the stability and the activity of the lactoferrin. He provides a method for purifying lactoferrin comprising the steps of contacting in a bind-and-elute mode and in an adsorptive fashion a solution of lactoferrin, with a hydrophilic absorbent and with a hydrophobic with the presence of surfactant, both in the presence of an excluded solute, and collecting a fraction containing lactoferrin substantially free of contaminant enzyme and/or lactoferrin inhibitor.
He has demonstrated that compared to the purified lactoferrin using its methods, the commercial lactoferrin manufactured from the same supplier as well as other suppliers available on the market did not display the same activity. In regards with the antibacterial activity, the purified lactoferrin did not lose its activity at higher concentration of lactoferrin in the medium (FIG. 4). None of the commercial lactoferrin preparations available on the market were able to display a minimum inhibition concentration and he has demonstrated that these commercial lactoferrin extracted from milk or whey have lost its, growth inhibitory activity at high concentration. Nevertheless, Dr Petitclerc has concluded that such phenomenon was due to the presence of proteases or degraded peptides Lf but he has never mentioned the presence of angiogenin.
Despite all the studies, none of the industrial processes, nor any other existing process for commercial scale purification, are able to purify the lactoferrin as it is present in our different secretion liquids.